Summary Our long term goal is to understand the mechanisms by which the spectrin-actin cytoskeleton supports dendritic arbor structure and function, and how these mechanisms are disrupted by Spinocerebellar Ataxia Type 5 (SCA5) mutations. SCA5 is a human neurodegenerative disease that causes gait and limb ataxia, slurred speech, and abnormal eye movements. The neuronal population targeted in SCA5 pathogenesis is cerebellar Purkinje cells. The disease stems from autosomal dominant mutations in the SPTBN2 gene encoding ?-III-spectrin. ?-III- spectrin is a key protein of the spectrin-actin cytoskeleton that localizes to Purkinje cell dendrites, and is required for normal dendritic arborization and signaling. We previously discovered that a SCA5 L253P mutation in the ?- III-spectrin actin-binding domain (ABD) causes a 1000-fold increase in actin affinity. In this project, we will test lead hypotheses for the molecular consequences of twelve additional mutations reported in SCA5 patients. A critical question we will address is whether SCA5 mutations in different domains of ?-III-spectrin converge on a common molecular consequence ? increased actin affinity. Thus, insights gained by the proposed mechanistic studies have the potential to provide a critical focus for the development of a SCA5 therapeutic. We will also test the alternative hypothesis that SCA5 mutations disrupt heterodimerization between ?- and ?-spectrin. Our prior cryo-EM studies led to the discovery of a novel ?-III-spectrin N-terminal domain that is required for actin binding in vitro. In this project, capitalizing on our expertise in whole-organismal studies using Drosophila, we will test if this domain is required for ?-spectrin function in vivo. Moreover, we will test if the N-terminal domain itself is regulated to control actin binding. We previously reported that a SCA5 mutation reduces the localization of spectrin and its binding partner, ankyrin-2, in dendrites of Drosophila neurons. However, the mechanisms by which spectrin and ankyrin work together to support dendrite structure and function are poorly understood. In this project, we will test if spectrin and ankyrin are required in dendrites for the organization of membrane proteins including ion channels and cell adhesion molecules. Thus these studies have the potential to inform on the mechanisms underlying SCA5-induced dendritic defects. The proposed studies will be successfully executed by a research team composed predominately of talented undergraduate students. The unique combination of molecular and whole-organismal studies will impassion students to pursue a career in the biomedical sciences.